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Chi-Chin Sun, Hsiao-Ting Chiu, Kuo-Ying Lee, Yi-Fang Lin, Jong Hwei Pang Su; Enhancement On Limbal Epithelial Cell Proliferation In Vitro By A ROCK Inhibitor, Y27632. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3512.
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© ARVO (1962-2015); The Authors (2016-present)
Transplantation of cultivated limbal progenitor cells is effective in restoring limbal stem cell deficiency. Recently, Rho kinase (ROCK) inhibitor Y27632 has been shown to improve corneal endothelial cell survival. The present study was conducted to investigate the applicability of Y27632 in promoting limbal epithelial cell proliferation in vitro.
Corneoscleral buttons from human donor eyes were serially expanded on culture dishes without or with Y27632 at various concentrations. The morphology and proliferation of limbal epithelial cells were monitored by a phase contrast microscopy and a luminescent cell-viability assay, respectively. Colony forming efficacy was detected by trypan blue staining. Proliferating cells were detected by Ki67 and p63 expression using immunofluorescent staining. Apoptotic limbal epithelial cells were analyzed by Annexin V staining.
The proliferation of expanded limbal epithelial cells was enhanced by Y27632 addition in a dose-dependent manner after 24 hours in culture. The colony forming efficacy of the culture was higher in the presence of Y27632 on day 7 in a dose-dependent manner. The number of p63 and Ki67-positive cells was dose-dependently increased at 3 weeks in Y27632-treated cultures. The number of Annexin V-positive apoptotic limbal epithelial cells was significantly decreased in Y27632-treated cultures at 3 weeks.
These results demonstrated that inhibition of ROCK signaling by Y27632 promoted limbal epithelial cell proliferation, increased the number of proliferating cells and inhibited apoptosis. It implies that the ROCK inhibitor may act as a new target for treating limbal stem cell deficiency.
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